LOCUS       sequence-489592-        6989 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Encodes wildtype human kindlin1 and N-terminal EBFP2 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  AUTHORS   Lee WN, Li J, Chen NP, Yu CH
  TITLE     Kindlin Regulates Mechanosensitive Activation and Adhesion Assembly 
            of Integrin beta6.
  JOURNAL   Adv Sci (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078.
  PUBMED    40492509
REFERENCE   2  (bases 1 to 6989)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Adv Sci 
            (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078."
COMMENT     Sequence Label: EBFP2-Kindlin1
FEATURES             Location/Qualifiers
     source          1..6989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     491..510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        765..1068
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1069..1272
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1223..1243
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1311..1320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1317..1319,1320..1322,1323..2033)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1362..1383)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4481..4602
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4518..4537)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4572..4591
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4609..5064)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4746..4767)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4958..4977
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5091..5195
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5197..5554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5197..5388
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5220..5240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5405..5540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5589..6383
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5589..6383
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5643..5662)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6253..6272
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6571..6590)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6615..6662
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
       61 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      121 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      661 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
      721 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      781 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      841 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      901 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      961 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1021 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1081 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1141 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1201 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1261 ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc gccaccatgg
     1321 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1381 acgtaaacgg ccacaagttc agcgtgaggg gcgagggcga gggcgatgcc accaacggca
     1441 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1501 tgaccaccct gagccacggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1561 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1621 aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctagtga
     1681 accgcatcga gctgaagggc gtcgacttca aggaggacgg caacatcctg gggcacaagc
     1741 tggagtacaa cttcaacagc cacaacatct atatcatggc cgtcaagcag aagaacggca
     1801 tcaaggtgaa cttcaagatc cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc
     1861 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac agccactacc
     1921 tgagcaccca gtccgtgctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1981 tggagttccg caccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     2041 tcagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc cctcgaccca
     2101 cgcgtccggc ggcgcgaggc ttcgctttcc tccagagcag cgacgcgggt gccgccagcc
     2161 tcgcctgttg ccgcgggagc ccggcgggag gagcgcggaa gggagccatg ctgtcatcca
     2221 ctgactttac atttgcttcc tgggagcttg tggtccgcgt tgaccatccc aatgaagagc
     2281 agcagaaaga cgtcacactg agagtatctg gagaccttca cgttggagga gtgatgctca
     2341 agttagtaga acagatcaat atatcccaag actggtcaga ctttgctctt tggtgggaac
     2401 agaagcattg ctggcttctg aaaacccact ggaccctgga caaatatggg gtccaggcag
     2461 atgcaaagct tctcttcacc cctcagcata aaatgctgcg ccttcgtctg ccgaatttga
     2521 agatggtgag gttgcgagtc agcttctcag ctgtggtttt taaagctgtc agtgatatct
     2581 gcaaaatcct gaatattaga agatcagaag agctttcctt gttaaagccg tctggtgact
     2641 attttaagaa gaagaagaaa aaagacaaaa ataataagga acccataatt gaagatattc
     2701 taaacctgga gagttctcca acagcttcag gttcatcagt aagtcctggt ttatacagta
     2761 aaaccatgac ccctatatat gaccccatca atggaacacc agcatcatcc accatgactt
     2821 ggttcagtga cagccctttg acggaacaaa actgcagcat cctcgcattc agccaacccc
     2881 cccagtcccc agaagcactt gcggatatgt accagcctcg gtctctggtt gataaagcca
     2941 agctcaatgc aggttggcta gactcctcac gctcccttat ggaacaaggc atccaagagg
     3001 atgagcagct gctcttacga tttaaatatt attctttctt cgacttgaat cctaaatatg
     3061 atgctgtccg aataaaccaa ctctatgagc aagccaggtg ggccattctc ttagaagaaa
     3121 ttgattgcac agaggaagaa atgttgatct ttgcagctct acagtaccac attagcaaac
     3181 tgtcgttgtc tgctgaaaca caggattttg caggcgagtc cgaggttgat gaaatagaag
     3241 cggcgctttc taatttggaa gtaaccctag aaggtggaaa agcggacagc cttttggagg
     3301 acattactga tatccctaaa cttgcagata atctcaaatt atttaggccc aagaagttac
     3361 taccaaaagc tttcaaacaa tattggttta tctttaaaga cacatccata gcatacttta
     3421 aaaataagga acttgaacaa ggagaaccac tagaaaaact aaatcttaga ggctgcgaag
     3481 ttgtgcccga tgtaaatgta gcaggaagaa aatttggaat caagttacta atccctgttg
     3541 ccgatggtat gaatgaaatg tatttgagat gtgaccatga gaatcaatac gcccaatgga
     3601 tggctgcctg catgttggca tcgaagggca aaaccatggc agacagctcc taccagccag
     3661 aggtcctcaa catcctttca tttctgagga tgaaaaacag gaactctgca tctcaggtgg
     3721 cttccagtct cgaaaacatg gatatgaacc cagaatgttt tgtgtcacca cggtgtgcaa
     3781 aaagacacaa atccaaacag ctggccgccc ggatcctgga ggcgcaccag aacgtggccc
     3841 agatgcccct ggtcgaagcc aagctgcggt tcatccaggc gtggcagtca ctgcctgagt
     3901 ttggcctcac ctactacctt gtcagattta aaggaagcaa aaaagatgac attctgggag
     3961 tttcatataa caggttgatt aaaattgatg cagccaccgg gattccagtg acaacatgga
     4021 gattcacaaa tatcaaacag tggaatgtaa actgggaaac ccggcaggtg gtcatcgagt
     4081 ttgaccaaaa cgtctttact gctttcacct gcctgagtgc agattgcaag attgtgcacg
     4141 agtacattgg cggctacatt ttcttgtcca cccgctccaa ggaccagaat gaaacactcg
     4201 atgaggactt gttccacaaa ttgaccggcg gtcaggattg aataggaaaa cttgtaataa
     4261 aactccacag ccataacaat atttaacttt aaagctattg tttcttatat gctgcttaat
     4321 aaagtaagct tgaactttaa aaaaaaaaaa aaagggcggc cgctctagat aactgatcat
     4381 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     4441 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     4501 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4561 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg
     4621 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4681 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4741 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4801 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4861 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4921 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4981 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5041 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5101 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5161 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     5221 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5281 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     5341 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5401 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5461 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5521 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     5581 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5641 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     5701 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5761 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5821 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5881 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5941 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     6001 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     6061 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     6121 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6181 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6241 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6301 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6361 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     6421 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6481 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     6541 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     6601 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     6661 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     6721 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     6781 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     6841 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6901 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6961 agaccccgta gaaaagatca aaggatctt
//
